The growing impact of proteins as efficacious drugs and diagnostic biomarkers is forcing the analytical community to deal with extremely high levels of analyte and sample complexity. However, due to the time and cost associated with developing an enzyme linked immunoassay (ELISA), mass spectrometric approaches are playing an increasing role in protein analyses. Furthermore, mass spectrometric methods enable detection of isoform variations and post-translational modifications; identification of these features being key to an understanding of protein function and activity.
Applications of enzymatic catalyses in biotechnology are often limited by the activity, stability and specificity of the enzyme under desired operating conditions. As such, the discovery and/or development of enzymes with improved characteristics can be the impetus for scientific breakthrough. Notably, Thermus aquaticus (Taq) polymerase has become one of the most important enzymes in molecular biology due to its ability to withstand the protein-denaturing conditions (high temperature) required during polymerase chain reaction (PCR) temperature cycling. Attempts to apply such enzymes to proteomics have resulted in restricted success. Thermolysin is an example of one such thermophilic enzyme applied to proteomics (Bark, et. al. J. Am. Chem. Soc. 2001, 123, 1774-1775). However, thermolysin cleaves hydrophobic amino acids and therefore lacks the specificity requisite for broad application. Alternatively trypsin is particularly well suited for mass spectrometric applications, since it has a very well defined specificity; it hydrolyzes only the peptide bonds in which the carbonyl group consists of either an arginine or lysine residue. However, many enzymes of homeothermic origin, including trypsin, have an optimal operating temperature of approximately 37° C. (Promega Technical Manual #9PIV511). As such, samples digested with these enzymes must be pretreated; proceeded by offline denaturation (e.g. guanidine, urea treatments), reduction (e.g. dithiothreitol, tris(2-carboxyethyl)phosphine treatments) and alkylation (e.g. n-ethylmaleimide, iodoacetamide, iodoacetic acid treatments). These steps are necessary for the exposure of cleavage sites existing within a protein's three dimensional structure. A thermostable form of trypsin, maintaining activity, stability and specificity at protein denaturing conditions would remove the need to perform these pretreatment steps.
In addition to a lack of thermal stability, another major limitation for many enzymes related to protein digestion pathways, including trypsin, is their low Kcat/Km ratio (catalytic efficiency). As such use of these enzymes is typically associated with hours-long incubation times. In and of itself, digestion is performed under kinetically unfavorable conditions. This phenomenon can be expressed using the Michaelis-Menten model (see FIG. 1). The drop in substrate concentration as a digestion nears completion makes it very difficult to obtain complete conversion to product. While enzymes typically produce product at an initial rate that is approximately linear for a short period after the start of the reaction, as the reaction proceeds and substrate is consumed, the rate continuously slows. The progression of substrate conversion to products can be represented as a process curve, as shown in FIG. 2. Using the digestion of a simple protein (e.g. insulin, see FIGS. 3 and 4) and a typical digestion protocol (50:1 protein:enzyme ratio, 37° C.) as an example it is easy to replicate this phenomenon in-situ. Incomplete trypsin digestion can lead to underestimations of protein concentrations. Furthermore, if some samples are more difficult to digest than others, for example plasma samples varying in protein content, an incomplete digestion would result in irreproducibility.
An enzyme working system that would eliminate the need to perform sample pretreatment while enabling a complete and reproducible digestion of protein samples is desired.